An “antibody fragment”, as used herein, shall mean the portion of an antibody comprising an antigen-binding region or a variable region thereof. Exemplary antibody fragments are Fab, Fab′, F(ab′)2 and scFv fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. When an antibody is treated with pepsine, F(ab′)2 fragment having two antigen-binding sites which is still capable of forming a cross-linking is produced. The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from a hinge region of the antibody.
In order to modify the inherent properties of an antibody by increasing the antibody binding affinity, changing the antigenicity or preparing a double specific antibody, various methods for the production of antibodies and fragments thereof using a gene recombinant technique have been studied. As a result, a method for producing an antibody and fragments thereof using an E. coli expression system has been developed. The production method using an E. coli expression system has several advantages over other conventional methods based on an animal cell expression system: 1) since it is easy to construct an expression vector, the expression thereof can be easily checked; 2) it is possible to mass-produce an antibody at a low cost because of the fast growth rate of E. coli, which facilitates securing antibody samples for experimental studies; and 3) application of the relatively simple fermentation method may allow an easier commercialization than those methods employing other host cells.
There have been several reports that an antibody-encoding gene is introduced into E. coli and expressed therein (Cabilly et al., Proc. Natl. Acad. Sci. USA 81: 3273-3277, 1984; and Boss et al., Nucleic Acids Res. 12: 3791-3806, 1984). These reports have disclosed that antibody molecules are expressed at various yields in a cytoplasmic space, and also that E. coli can be used as a host cell for secretively expressing an immunoglobulin light chain (Zemel-Dreasen et al., Gene 315-322, 1984), or for secreting an antibody fragment fused with alkaline phosphatase or β-lactamase signal sequence into a periplasmic space (Pluckthun et al., Cold Spring Harbor Symposiua on Quantitative Biology Volume LII, 105-112, 1987). International Patent Publication No. WO 92/01059 teaches that the Fab′-encoding gene which recognizes a specific site of a cancer cell is expressed in the form of a fusion protein with E. coli outer membrane protein A (OmpA) signal sequence; and U.S. Pat. No. 5,648,237 discloses that the Fab′-encoding gene fused with E. coli enterotoxin signal sequence is expressed under the control of PhoA promoter.
An expressed antibody fragment shows an antigen-binding affinity similar to a wild-type antibody; however, since it is smaller than the wild-type, its local invasiveness is higher (Blumenthal et al., Adv. Drug Del. Rev. 4: 279, 1990), and as it does not contain any Fc region, no side-effect is induced when administered to a human body. Accordingly, an antibody fragment may be used to prepare a diagnostic reagent or to develop a therapeutic antibody, and the mass-production thereof in E. coli becomes economically attractive. In case of preparing an antibody fragment from a wild-type antibody, the wild-type antibody must be digested with a proteinase and purified, which is cumbersome and economically unfavorable.
However, there are several problems associated with the production of an antibody fragment in E. coli. First, not all antibody fragments can be expressed in an amount sufficient for intended therapeutic or diagnostic purpose, and sometimes the cultivation procedure must be repeated several times. Further, to endow the antibody fragment with functional activity, both the heavy chain and the light chain have to be expressed in a single cell and maintain a heterozygote form via a disulfide bond formed between them, which requires that the antibody fragment must be expressed in a highly soluble form, and not in the form of an inclusion body which is frequently found when a protein is overly expressed in E. coli. To solve such problems, a method for secreting an expressed antibody fragment into a periplasmic space using a signal sequence has been actively studied. However, it has been reported that not all antibody fragments fused with the signal sequence are secretively over-expressed into the periplasmic space of E. coli, and the amount of the expressed antibody varies greatly with the nucleotide sequence thereof (Kelly et, al., Biochemistry 31: 5434-5441, 1992; and Humphreys et, al., Protein Expression and Purification 26: 309-320, 2002). It has also been found that the expression pattern of an antibody fragment fluctuates depending on the difference in the expression vector's structural gene or the distance between the genes encoding the heavy chain and the light chain (International Patent Publication No: WO 01/94585). These facts suggest that for the expression of a target antibody fragment, a proper balance must be maintained in terms of the interaction between the target antibody gene and the host structural gene essential for the expression and the interaction between the target and signal sequences in case signal sequences are used.
The present inventors have therefore endeavored to develop a method for expressing in E. coli an antibody fragment in a soluble heterolzygote form having a desired antigen-binding affinity by preparing a recombinant expression vector which secretively expresses human antibody Fab′ of tumor necrosis factor-alpha (TNF-α) as a target antibody fragment using E. coli thermostable enterotoxin signal sequence or E. coli outer membrane protein A (Omp A) signal sequence.